PROJECT SUMMARY Influenza A virus is a negative-strand RNA virus and is classified into subtypes based on the antigenic properties of the two surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA). There are 18 known HA subtypes (H1 to H18) and 11 known NA subtypes (N1 to N11). Wild aquatic birds are the main natural reservoir for influenza A viruses. However, spillover from natural reservoirs can cause human pandemics. Three subtypes (H1N1, H2N2, and H3N2) have caused human pandemics, and two of them (H1N1 and H3N2) are presently circulating the in human population, causing seasonal outbreaks. Other subtypes found in natural reservoirs also occasionally transmit to the human population, as exemplified by H5N1, H5N6, H6N1, H7N7, H7N9, H9N2, and H10N8 viruses. These zoonotic subtypes can be highly pathogenic and have a high mortality rate (>50% of hospitalized individuals) when infecting humans. The virus- host protein-protein interaction constitutes the molecular basis of influenza pathogenicity. Many ?omics? studies have explored influenza-host interactions. However, the known mechanistic details are far from comprehensive, especially regarding variation in pathogenicity across different virus subtypes and hosts. The objective of the proposed study is to understand how genetic variation in influenza virus and the host together influence pathogenicity. This proposal will focus on influenza NS1 protein, which is a major pathogenicity determinant and plays a role in host adaptation. NS1 is known to interact with many host proteins. Some of these interactions are known to be influenza subtype-dependent. In the proposed study, NS1 from two seasonal subtypes (H1N1 and H3N2) and one zoonotic subtype (H5N1) will be employed. The difference in the functional constraints of these three NS1 in human and avian cells will be interrogated by deep mutational scanning and systematic interactome analysis (K99 phase). Subsequently, biophysical characterization of NS1-TRIM25 interaction will be performed. NS1 interacts with TRIM25, which is an E3 ubiquitin ligase, to suppress the innate immune response. NS1-TRIM25 interaction is virus subtype-dependent and host-dependent. The underlying biophysical mechanism will be probed by affinity maturation and cryo- electron microscopy (K99/R00 phase). The proposed study will facilitate a detailed understanding of influenza pathogenicity and tropism from the molecular biology, genetics, evolutionary, and structural biology perspectives. More importantly, the integrative research framework developed in this study will be applicable to other influenza virus proteins, or even to proteins from other viruses.